The cell membrane (also known as the plasma membrane or cytoplasmic membrane) is a biological membrane that separates the interior of the cell from the outside environment, protecting the cell from its surroundings. The membrane comprises a phospholipid bilayer with embedded proteins, and is involved in cellular processes such as cell adhesion, ion conductivity and cell signaling.
The cell membrane controls the movement of substances in and out of cells and is selectively permeable to ions and organic molecules. The movement of substances across the membrane may be passive (i.e., occurring without the input of cellular energy) or active (i.e., requiring the cell to expend energy in transporting it). The cell membrane thus works as a selective filter, employing transport mechanisms such as passive osmosis and diffusion, transmembrane protein channels transportation, endocytosis and exocytosis.
Intracellular delivery of biologically active compounds is challenging because the cell membrane is remarkably impermeable to extracellular polar compounds. There is thus much interest in identifying novel cell-permeable peptides (“CPPs”) that can act as “Trojan horses” for carrying cargo molecules inside living cells. CPPs have been employed in intracellular delivery of oligonucleotides (Astriab-Fisher et al., 2000, Biochem. Pharmacol. 60:83-90; Eguchi et al., 2001, J. Biol. Chem. 276:26204-26210), plasmids (Morris et al., 1999, Nucleic Acids Res. 27:3510-3517), viruses (Gratton et al., 2003, Nat. Med. 9:357-362), peptides (Gratton et al., 2003, Nat. Med. 9:357-362; Soomets et al., 2000, Biochim. Biophys. Acta 1467:165-176) and fluorophores (Bucci et al., 2000, Nat. Med. 6:1362-1367). The Antennapedia homeodomain (“AP”; a 16-amino acid peptide, which is a Drosophila transcription factor), as well as the HIV transactivator of transcription (“TAT”; 15 amino acids) are amongst the first CPPs described, along with more recently described CPP sequences, such as poly-Arginine (Arg7 or Arg9) and C105Y (a 17-amino acid peptide).
The capacity of CPPs to translocate cargo into cells could make them attractive delivery agents for cell-impermeable therapeutic compounds. However, the therapeutic effect, kinetics, safety profile and specificity of CPPs in humans are still unknown. Novel target-engineered CPPs with enhanced internalization capabilities, enhanced overall therapeutic efficacy and safety, minimal peptide elimination/degradation and high therapeutic activity/cost ratio are required.
Caveolins are cholesterol binding proteins that may regulate signal transduction pathways (Smart et al., 1999, Mol. Cell. Biol. 19:7289-7304; Kurzchalia & Parton, 1999, Curr. Opin. Cell. Biol. 11:424-431). Recent studies have focused on their subcellular trafficking and regulation of endothelial nitric oxide synthase (eNOS). eNOS-derived NO is necessary for the maintenance of systemic blood pressure, vascular remodeling, angiogenesis and wound healing (Huang et al., 1995, Nature 377:239-242; Murohara et al., 1998, J. Clin. Invest. 101:2567-2578; Rudic et al., 1998, J. Clin. Invest. 101:731-736; Lee et al., 1999, Am. J. Physiol. 277:HI600-1608). eNOS can physically interact with caveolin-1 and caveolin-3 by binding to their putative scaffolding domain located between residues 82-101 (Li et al., 1996, J. Biol. Chem. 271:29182-29190), and this interaction renders eNOS in its “less active” state (Garcia-Cardena et al., 1997, J. Biol: Chem. 272:25437-25440; Ju et al., 1997, J. Biol. Chem. 272:18522-18525; Michel et al., 1997, J. Biol. Chem. 272:25907-25912). Consistent with the model of caveolin as a negative regulator of eNOS, peptides derived from the scaffolding domain of caveolin-1 disrupt the binding of eNOS to caveolin and inhibit NOS activity in a dose dependent manner in vitro (IC50=1-3 μM) by slowing electron flux from the reductase to the oxygenase domain of NOS (Garcia-Cardena et al., 1997, J. Biol. Chem. 272:25437-25440; Ju et al., 1997, J. Biol. Chem. 272:18522-18525; Ghosh et al., 1998, J. Biol. Chem. 273:22267-22271).
There is a need in the art to identify novel molecules that efficiently penetrate cell membranes. Such molecules would be useful in promoting the delivery of cargo moieties, such as therapeutic agents, nucleic acids, peptides, saccharides, lipids, liposomes and such, across the cell membrane. The present invention satisfies this unmet need.